Henk E. Moorlag

A Novel Strategy to Modify Adenovirus Tropism and Enhance Transgene Delivery to Activated Vascular Endothelial Cells In Vitro and In Vivo

To assess the possibilities of retargeting adenovirus to activated endothelial cells, we conjugated bifunctional polyethylene glycol (PEG) onto the adenoviral capsid to inhibit the interaction between viral knob and coxsackie-adenovirus receptor (CAR). Subsequently, we introduced an alphav integrin-specific RGD peptide or E-selectin-specific antibody to the other functional group of the PEG molecule for the retargeting of the adenovirus to activated endothelial cells. In vitro studies showed that this approach resulted in the elimination of transgene transfer into CAR-positive cells, while at the same time specific transgene transfer to activated endothelial cells was achieved. PEGylated, retargeted adenovirus showed longer persistence in the blood circulation with area under plasma concentration-time curve (AUC) values increasing 12-fold compared to unmodified virus. Anti-E-selectin antibody-PEG-adenovirus selectively homed to inflamed skin in mice with a delayed-type hypersensitivity (DTH) inflammation, resulting in local expression of the reporter transgene luciferase. This is the first study showing the benefits of PEGylation on adenovirus behavior upon systemic administration. The approach described here can form the basis for further development of adenoviral gene therapy vectors with improved pharmacokinetics and increased efficiency and specificity of therapeutic gene transfer into endothelial cells in disease.

Targeting of RGD-modified proteins to tumor vasculature : A pharmacokinetic and cellular distribution study

Angiogenesis‐associated integrin αvβ3 represents an attractive target for therapeutic intervention because it becomes highly upregulated on angiogenic endothelium and plays an important role in the survival of endothelial cells. Cyclic RGD peptides were prior shown to have a high affinity for αvβ3 and can induce apoptosis of endothelial cells. In our laboratory, monocyclic RGD peptides (cRGDfK) were chemically coupled to a protein backbone. Previous results demonstrated that the resulting RGDpep‐HuMab conjugate bound with increased avidity to αvβ3/αvβ5 on endothelial cells. In our present study, RGDpep‐HuMab was injected intravenously and intraperitoneally in B16.F10 tumor‐bearing mice to determine its pharmacokinetics and organ distribution. In the tumor, the RGDpep‐HuMab conjugate specifically localized at the endothelium as was demonstrated by immunohistochemistry. The control RADpep‐HuMab conjugate was not detected in the tumor. Besides tumor localization RGDpep‐HuMab was found in liver and spleen associated with macrophages. This uptake by macrophages is probably responsible for the more rapid clearance of RGDpep‐HuMab from the circulation than HuMab and RADpep‐HuMab. The half‐life of RGDpep‐HuMab (90 min) was still considerably longer than that of free RGD peptides (<10 min). This prolonged circulation time may be favorable for drug targeting strategies because the target cells are exposed to the conjugate for a longer time period. Taken together these results indicate that RGD‐modified proteins are suitable carriers to deliver therapeutic agents into tumor or inflammation induced angiogenic endothelial cells.

Endothelial cells internalize and degrade RGD-modified proteins developed for tumor vasculature targeting

Tumor vasculature can be targeted by peptides containing an RGD (Arg-Gly-Asp) sequence, which bind to alpha(v)beta3 and alpha(v)beta5 integrins on angiogenic endothelial cells. By covalently attaching cyclic RGD-peptides (cRGDfK) to a protein backbone, we prepared a multivalent peptide-protein conjugate with increased affinity for alpha(v)beta3/alpha(v)beta5 integrins. We demonstrated that RGDpep-protein conjugate bound to HUVEC, whereas the conjugate prepared with the control RAD peptide was devoid of any binding. RGDpep-protein conjugate was furthermore functional in inhibiting the adhesion of HUVEC to alpha(v)beta3/alpha(v)beta5 ligand vitronectin, and direct binding of the radiolabeled conjugate to HUVEC was inhibited by alpha(v)beta(3)/alpha(v)beta5-specific RGD peptides. Finally, RGDpep-protein conjugate was shown to be internalized and degraded by HUVEC, a process that could be inhibited by lysosomal degradation inhibitors chloroquine and ammonium chloride. This cellular handling was significantly influenced by the presence of cations, which strongly inhibited internalization. This is the first study that shows direct evidence that primary endothelial cells are capable of internalizing RGD-containing macromolecular proteins. This feature makes them attractive carriers for the intracellular delivery of potent anti-angiogenic drugs into endothelial cells for the treatment of cancer and chronic inflammatory diseases.

Angiogenesis in Synchronous and Metachronous Colorectal Liver Metastases The Liver as a Permissive Soil

Objective:Resection of a primary colorectal carcinoma (CRC) can be accompanied by rapid outgrowth of liver metastases, suggesting a role for angiogenesis. The aim of this study is to investigate whether the presence of a primary CRC is associated with changes in angiogenic status and proliferation/apoptotic rate in synchronous liver metastases and/or adjacent liver parenchyma. Methods:Gene expression and localization of CD31, HIF-1&agr;, members of the vascular endothelial growth factor (VEGF) and Angiopoietin (Ang) system were studied using qRT-PCR and immunohistochemistry in colorectal liver metastases and nontumorous-adjacent liver parenchyma. Proliferation and apoptotic rate were quantified. Three groups of patients were included: (1) simultaneous resection of synchronous liver metastases and primary tumor (SS-group), (2) resection of synchronous liver metastases 3 to 12 months after resection of the primary tumor [late synchronous (LS-group)], and (3) resection of metachronous metastases >14 months after resection of the primary tumor (M-group). Results:In all 3 groups a higher expression of the angiogenic factors was encountered in adjacent liver parenchyma as compared to the metastases. VEGFR-2 gene expression was abundant in adjacent liver parenchyma in all 3 groups. VEGF-A and VEGFR-1 were prominent in adjacent parenchyma in the SS-group. The SS-group showed the highest Ang-2/Ang-1 ratio both in the metastases and the adjacent liver. This was accompanied by a high turnover of tumor cells. CONCLUSIONIn the presence of the primary tumor, the liver parenchyma adjacent to the synchronous liver metastases provides an angiogenic prosperous environment for metastatic tumor growth.

Neovascularization and vascular markers in a foreign body reaction to subcutaneously implanted degradable biomaterial in mice

To study the spatiotemporal processes of angiogenesis during a foreign body reaction (FBR), biodegradable bovine collagen type-1 (COL-I) discs were implanted in mice for a period up to 28 days. The cellular infiltration (consisting mainly of macrophages, giant cells and fibroblasts), and the extent of neovascularization into the discs were determined. Also the expression levels and/or distribution of the endothelial cell markers von Willebrand factor (vWF), platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, MECA-32 antigens and endomucin, and of the basal lamina marker collagen type IV (Coll IV) were analysed. In time, a strong neovascularization of the discs was observed, with frequently occurring vascular sprouting, and intussusceptive growth of vessels. In this model, vWF, MECA-32 and endomucin antibodies often failed to stain neovessels in the COL-I discs. In contrast, staining for collagen IV basal lamina component in combination with CD31 covered the complete range of neo-vessels. We conclude that the model described in this study is a useful model to study FBR induced angiogenesis because of the active neovascularization taking place during prolonged periods of time.

Interactions between Blood-Borne Streptococcus pneumoniae and the Blood-Brain Barrier Preceding Meningitis

Streptococcus pneumoniae (the pneumococcus) is a Gram-positive bacterium and the predominant cause of bacterial meningitis. Meningitis is thought to occur as the result of pneumococci crossing the blood-brain barrier to invade the Central Nervous System (CNS); yet little is known about the steps preceding immediate disease development. To study the interactions between pneumococci and the vascular endothelium of the blood-brain barrier prior to meningitis we used an established bacteremia-derived meningitis model in combination with immunofluorescent imaging. Brain tissue of mice infected with S. pneumoniae strain TIGR4, a clinical meningitis isolate, was investigated for the location of the bacteria in relation to the brain vasculature in various compartments. We observed that S. pneumoniae adhered preferentially to the subarachnoid vessels, and subsequently, over time, reached the more internal cerebral areas including the cerebral cortex, septum, and choroid plexus. Interestingly, pneumococci were not detected in the choroid plexus till 8 hours-post infection. In contrast to the lungs, little to no leukocyte recruitment to the brain was observed over time, though Iba-1 and GFAP staining showed that microglia and astrocytes were activated as soon as 1 hour post-infection. Our results imply that i) the local immune system of the brain is activated immediately upon entry of bacteria into the bloodstream and that ii) adhesion to the blood brain barrier is spatiotemporally controlled at different sites throughout the brain. These results provide new information on these two important steps towards the development of pneumococcal meningitis.

PLE catalyzed hydrolyses of α-substituted α-hydroxy esters: The influence of the substituents.

Abstract The enzymatic hydrolyses of a variety of α-substituted mandelic and lactic esters using pig liver esterase (PLE) have been investigated. High to moderate enantioselectivity was found for various α-substituted mandelic esters, whereas PLE showed low to no enantioselectivity for α-substituted lactic esters. We observed that the enantioselectivity of PLE depends strongly on the length and nature of the substituent at the α-position. Some consequences for an active site model of PLE are discussed.

Vascular endothelial growth factor receptor 2 inhibition in-vivo affects tumor vasculature in a tumor type-dependent way and downregulates vascular endothelial growth factor receptor 2 protein without a prominent role for miR-296

The precise molecular effects that antiangiogenic drugs exert on tumor vasculature remain to be poorly understood. We therefore set out to investigate the molecular and architectural changes that occur in the vasculature of two different tumor types that both respond to vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor therapy. Mice bearing Lewis lung carcinoma (LLC) or B16.F10 melanoma were treated with vandetanib (ZD6474), a VEGFR2/epidermal growth factor receptor (EGFR)/REarranged during Transfection (RET) kinase inhibitor, resulting in a significant 80% reduction in tumor outgrowth. Although in LLC the vascular density was not affected by vandetanib treatment, it was significantly decreased in B16.F10. In LLC, vandetanib treatment induced a shift in vascular gene expression toward stabilization, as demonstrated by upregulation of Tie2 and N-cadherin and downregulation of Ang2 and integrin &bgr;3. In contrast, only eNOS and P-selectin responded to vandetanib treatment in B16.F10 vasculature. Strikingly, vandetanib reduced protein expression of VEGFR2 in both models, whereas mRNA remained unaffected. Analysis of miR-296 expression allowed us to exclude a role for the recently proposed microRNA-296 in VEGFR2 posttranslational control in LLC and B16.F10 in vivo. Our data demonstrate that VEGFR2/EGFR inhibition through vandetanib slows down both LLC and B16.F10 tumor growth. Yet, the underlying molecular changes in the vasculature that orchestrate the antitumor effect differ between tumor types. Importantly, in both models, vandetanib treatment induced loss of its pharmacological target, which was not directly related to miR-296 expression. Validation of our observations in tumor biopsies from VEGFR2 inhibitor-treated patients will be essential to unravel the effects of VEGFR2 inhibitor therapy on tumor vasculature in relation to therapeutic efficacy.

Histone Deacetylase Inhibition and IκB Kinase/Nuclear Factor-κB Blockade Ameliorate Microvascular Proinflammatory Responses Associated With Hemorrhagic Shock/Resuscitation in Mice.

Objective:To investigate the consequences of histone deacetylase inhibition by histone deacetylase inhibitor valproic acid and I&kgr;B kinase/nuclear factor-&kgr;B signaling blockade by I&kgr;B kinase inhibitor BAY11-7082 on (microvascular) endothelial cell behavior in vitro as well as in mice subjected to hemorrhagic shock/resuscitation in vivo. Design:Prospective, randomized laboratory investigation using an established mouse model of hemorrhagic shock. Setting:Research laboratory at university teaching hospital. Subjects:Endothelial cells and C57BL/6 male mice. Interventions:Endothelial cells were incubated with tumor necrosis factor-&agr; in the absence or presence of valproic acid or BAY11-7082 in vitro. Mice were subjected to hemorrhagic shock by blood withdrawn until the mean arterial pressure of 30 mm Hg and maintained at this pressure for 90 minutes. At 90 minutes, subgroups of mice were resuscitated with 4% human albumin in the absence or presence of vehicle, valproic acid (300 &mgr;g/g body weight) or BAY11-7082 (400 &mgr;g per mouse). Mice were killed 1 hour and 4 hours after resuscitation. Measurements and Main Results:Valproic acid and BAY11-7082 selectively diminished tumor necrosis factor-&agr;-induced endothelial proinflammatory activation in vitro. In vivo, both systemic and local inflammatory responses were significantly induced by hemorrhagic shock/resuscitation. The decreased histone acetylation in kidneys after hemorrhagic shock/resuscitation was restored by valproic acid treatment. In glomerular endothelial cells, the nuclear translocation of nuclear factor-&kgr;B, which was induced by hemorrhagic shock/resuscitation, was eliminated by BAY11-7082 treatment while enhanced in the presence of valproic acid. Both valproic acid and BAY11-7082 significantly attenuated the hemorrhagic shock/resuscitation-induced protein expression of endothelial cell adhesion molecules E-selectin and vascular cell adhesion molecule-1 in the microvasculature of kidneys and liver, although messenger RNA expression levels of these molecules analyzed in whole-organ lysates of kidneys, lungs, and liver were not extensively affected. The reduced protein expression of adhesion molecules was paralleled by diminishing the adhesion/transmigration of neutrophils in kidneys and liver after hemorrhagic shock/resuscitation. Conclusion:Suppression of histone deacetylase activity and blockade of I&kgr;B kinase/nuclear factor-&kgr;B signaling during resuscitation ameliorate microvascular endothelial proinflammatory responses in organs in mice after hemorrhagic shock.

Tumor Vascular Morphology Undergoes Dramatic Changes during Outgrowth of B16 Melanoma While Proangiogenic Gene Expression Remains Unchanged

In established tumors, angiogenic endothelial cells (ECs) coexist next to “quiescent” EC in matured vessels. We hypothesized that angio-gene expression of B16.F10 melanoma would differ depending on the growth stage. Unraveling the spatiotemporal nature thereof is essential for drug regimen design aimed to affect multiple neovascularization stages. We determined the angiogenic phenotype—represented by 52 angio-genes—and vascular morphology of small, intermediate, and large s.c. growing mouse B16.F10 tumors and demonstrated that expression of these genes did not differ between the different growth stages. Yet vascular morphology changed dramatically from small vessels without lumen in small to larger vessels with increased lumen size in intermediate/large tumors. Separate analysis of these vascular morphologies revealed a significant difference in αSMA expression in relation to vessel morphology, while no relation with VEGF, HIF-1α, nor Dll4 expression levels was observed. We conclude that the tumor vasculature remains actively engaged in angiogenesis during B16.F10 melanoma outgrowth and that the major change in tumor vascular morphology does not follow molecular concepts generated in other angiogenesis models.